Bulek, A. | Search bioRxiv

bioRxiv2023

Designer small molecule control system based on Minocycline induced disruption of protein-protein interaction.

A versatile, safe, and effective small-molecule control system is highly desirable for clinical cell therapy applications. Therefore, we developed a two-component small-molecule control system based on the disruption of protein-protein interactions using minocycline, an FDA-approved antibiotic with wide availability, excellent bio-distribution, and low toxicity. The system comprises an anti-minocycline single-domain antibody (sdAb) and a minocycline-displaceable cyclic peptide. Here we show how this versatile system can be applied to OFF-switch split CAR systems (MinoCAR) and universal CAR adaptors (MinoUniCAR) with reversible, transient, and dose-dependent suppression; to a tunable T cell activation module based on MyD88/CD40 signaling; to a controllable cellular payload secretion system based on IL-12 KDEL retention and as a cell/cell inducible junction. This work represents an important step forward in the development of a remote-controlled system to precisely control the timing, intensity, and safety of therapeutic interventions. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=125 SRC="FIGDIR/small/553207v1_ufig1.gif" ALT="Figure 1"> View larger version (57K): org.highwire.dtl.DTLVardef@1a16d73org.highwire.dtl.DTLVardef@1189ceeorg.highwire.dtl.DTLVardef@e70a57org.highwire.dtl.DTLVardef@580fa9_HPS_FORMAT_FIGEXP M_FIG C_FIG

molecular biology↗

bioRxiv2023

Exploration of T-cell immune responses by expression of a dominant-negative SHP1 and SHP2

SHP1 and SHP2 are SH2 domain-containing proteins which have inhibitory phosphatase activity when recruited to phosphorylated ITIMs and ITSMs on inhibitory immune receptors. Consequently, SHP1 and SHP2 are key proteins in the transmission of inhibitory signals within T cells, constituting an important point of convergence for diverse inhibitory receptors. Therefore, SHP1 and SHP2 inhibition may represent a strategy for preventing immunosuppression of T cells mediated by cancers hence improving immunotherapies directed against these malignancies. Both SHP1 and SHP2 contain dual SH2 domains responsible for localization to the endodomain of inhibitory receptors and a protein tyrosine phosphatase domain which dephosphorylates and thus inhibits key mediators of T cell activation. We explored the interaction of the isolated SH2 domains of SHP1 and SHP2 to inhibitory motifs from PD1 and identified strong binding of both SH2 domains from SHP2 and more moderate binding in the case of SHP1. We next explored whether these a truncated form of SHP1/2 comprising only of SH2 domains (dSHP1/2) could act in a dominant negative fashion by preventing docking of the wild type proteins. When co-expressed with CARs we found that dSHP2 but not dSHP1 could alleviate immunosuppression mediated by PD1. We next explored the capacity of dSHP2 to bind with other inhibitory receptors and observed several potential interactions. In vivo we observed that the expression of PDL1 on tumor cells impaired the ability of CAR T cells to mediate tumor rejection and this effect was partially reversed by the co-expression of dSHP2 albeit at the cost of reduced CAR T cell proliferation. Modulation of SHP1 and SHP2 activity in engineered T cells through the expression of these truncated variants may enhance T cell activity and hence efficacy in the context of cancer immunotherapy.

immunology↗